Abstract
Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74-282)) and the conserved C-terminal region (p32 (1-221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.
| Original language | English |
|---|---|
| Pages (from-to) | 771-781 |
| Number of pages | 11 |
| Journal | Cell Death and Differentiation |
| Volume | 11 |
| Issue number | 7 |
| DOIs | |
| Publication status | Published - Jul 1 2004 |
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All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology
Cite this
Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32. / Sunayama, J.; Ando, Y.; Itoh, N.; Tomiyama, A.; Sakurada, K.; Sugiyama, A.; Kang, Dongchon; Tashiro, F.; Gotoh, Y.; Kuchino, Y.; Kitanaka, C.
In: Cell Death and Differentiation, Vol. 11, No. 7, 01.07.2004, p. 771-781.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32
AU - Sunayama, J.
AU - Ando, Y.
AU - Itoh, N.
AU - Tomiyama, A.
AU - Sakurada, K.
AU - Sugiyama, A.
AU - Kang, Dongchon
AU - Tashiro, F.
AU - Gotoh, Y.
AU - Kuchino, Y.
AU - Kitanaka, C.
PY - 2004/7/1
Y1 - 2004/7/1
N2 - Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74-282)) and the conserved C-terminal region (p32 (1-221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.
AB - Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74-282)) and the conserved C-terminal region (p32 (1-221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.
UR - http://www.scopus.com/inward/record.url?scp=3543075659&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=3543075659&partnerID=8YFLogxK
U2 - 10.1038/sj.cdd.4401418
DO - 10.1038/sj.cdd.4401418
M3 - Article
C2 - 15031724
AN - SCOPUS:3543075659
VL - 11
SP - 771
EP - 781
JO - Cell Death and Differentiation
JF - Cell Death and Differentiation
SN - 1350-9047
IS - 7
ER -